In this application we propose to continue our studies aimed on understanding the molecular basis of the pathogenic process in transmissible spongiform encephalopathies, a group of fatal neurodegenerative diseases such as Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. It is believed that the infectious pathogen responsible for these diseases is a misfolded, proteinase-resistant form of the prion protein, PrPSc, which is derived from a precursor PrPC by a poorly understood mechanism. While the structure of PrPC is well characterized, one of the most enduring mysteries in the field is the molecular structure of infectious PrPSc conformers. In this project, we propose to undertake a major effort to bridge the gap in understanding the structural properties of abnormally folded prion protein aggregates, with specific aims focused on: (i) determining the conformational structure of proteinase K- resistant form of the recombinant Syrian hamster prion protein, PrPres, obtained by brain PrPSc- templated protein misfolding cyclic amplification (PMCA) reaction; (ii) determining medium- resolution structure (site- and region-specific structural constraints) for brain-derived hamster PrPSc; (iii) elucidating the nature of structural differences between PrPSc corresponding to the hyper and drowsy strains of TSE. This study will employ a number of complementary biophysical methods including site-directed spin labeling, solid-state NMR spectroscopy, hydrogen/deuterium exchange, and hydroxyl radical-mediated footprinting coupled with mass spectrometry.